Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, E. C. 4.1.1.39) is the most abundant and perhaps most important enzyme on earth. It catalyzes the first and rate-limiting step in photosynthetic carbon fixation, the transfer of atmospheric CO2 to ribulose-1,5-bisphosphate. As such, it is the only known enzyme able to remove CO2 from the atmosphere. Because of its keystone position in biomass production, the importance of Rubisco to agriculture is hard to overstate. Cash receipts for American agricultural products in 1997 were $209 billion, of which $112 billion were earned directly from crops (Economic Research Service, USDA). Thus, any incremental increase in crop productivity will be leveraged through a huge sector of the US agricultural economy. For several reasons, it is widely supposed that increasing Rubisco's catalytic efficiency will result in a significant increase in plant productivity. First, the reaction catalyzed by Rubisco is rate limiting to plant growth under optimum growing conditions (high temperature and light intensity, abundant nitrogen). Second, compared to many other enzymes, Rubisco seems to be an inefficient catalyst that leaves a great deal of room to be optimized.
As a catalyst, Rubisco appears to be sub-optimal in three respects. First, its catalytic cycling rate (kcat) at about 3 reactions per second, for the enzymes from higher plants, is relatively slow. To compensate for its low activity, plants deposit large amounts of Rubisco enzyme in their green tissues. Indeed, Rubisco accounts for more than 35% of leaf total soluble proteins. Increasing Rubisco's catalytic efficiency would proportionally increase the rate of photosynthesis and, in turn, increase plant productivity. Second, Rubisco cannot effectively distinguish CO2 from O2 and, consequently, it catalyzes an oxygenation reaction that leads to the loss of approximately 25% to 40% of fixed carbon. Theoretically, it is possible to increase plant productivity up to 50% by reducing or eliminating Rubisco's oxygenase activity.
Rubisco has become one of the most intensively investigated plant enzymes. Evolution and adaptation of Rubisco in its various native hosts have resulted in a naturally occurring diversity of enzymatic properties (Jordan and Ogren, 1981). Compared to plant Rubisco, the enzyme from prokaryotic photosynthetic bacteria generally possesses higher catalytic activity (kcat≈8-16 s−1), but low CO2/O2 selectivity (τ≈13-40). τ is the ratio of kcat (carboxylation)/Km(CO2) over kcat (oxygenation)/Km (O2) (Laing, et al., 1974). Rubisco from higher plants including crop species exhibits low kcat (≈3 s−1), and an intermediate CO2/O2 selectivity (τ≈80). The recently-assayed Rubisco from red algae shows the highest CO2/O2 selectivity yet measured (τ≈140-300, Ezaki, et al., 1999; Read and Tabita, 1994; Uemura, et al., 1997), but the kcat assayed at 25° C. is lower than that of higher plant Rubisco. This diversity among Rubisco enzymes stimulated research aimed at understanding the structure/function relationships that account for the variation of the catalytic parameters kcat and τ. Engineering a better Rubisco through knowledge of the structural determinants of kcat and τ constitutes the so called “rational approach.”
Rubisco from different organisms displays different physical and chemical features. Its holoenzyme is a multi-subunit complex. The primitive form is a large/large subunit dimer (L2). The L2 enzyme is mainly present in anaerobic proteobacteria, but the L2 enzyme is also formed in some eukaryotic algae under anaerobic conditions. In all higher plants and cyanobacteria, Rubisco is composed of eight large (L) and eight small (S) subunits (L8S8). The L subunit is encoded by a chloroplast gene (rbcL), and the S subunit is encoded by a nuclear gene family (rbcS). So far, only L2, the cyanobacterial L8S8 enzyme, and an L8 enzyme from a hyperthermophilic alga have been expressed and assembled in E. coli. Expression of higher plant Rubisco L and S simultaneously in E. coli resulted in no holoenzyme being formed. Consequently, most Rubisco engineering research has been limited to prokaryotic enzymes and the enzyme from the eukaryotic algae Chlamydomonas reinhardtii. 
For more than 30 years a number of researchers have attempted to improve Rubisco, using a variety of approaches. See, e.g., Mann, C. C., (1999) Science, 283:314-316, and references cited therein. Indeed, the quest for a better Rubisco has been called a “Holy Grail” of plant biology. To date, there has been little success in the creation of an improved Rubisco. Recombination based methods for producing a modified Rubisco enzyme having increased catalytic efficiency and selectivity for CO2 are described in U.S. patent application Ser. No. 09/437,726.
An obstacle hindering the improvement of Rubisco is the deficiencies in currently available host systems for the expression and assembly of functional higher plant Rubisco. In screening a large number of variants for enhanced activity, preferred host systems have included E. coli, yeast, cyanobacteria and green algae. In the case of prokaryotic Rubisco, the large subunit (i.e., the L8 core) of prokaryotic Rubisco is soluble, and catalytically competent holoenzyme can be formed in E. coli with the help of a chaperone protein (GroEL) present in E. coli. In contrast, the large subunit from higher plant Rubisco is insoluble; this is thought to be caused by a hydrophobic surface that is protected by the small subunit in the holoenzyme. In chloroplasts, assembly of the large subunits with mature small subunits is mediated by a chaperone protein, Rubisco binding protein (cpn60). The chaperone protein is believed to prevent improper aggregation of large subunits by protecting exposed hydrophobic surfaces during the last stages of the folding or assembly process. Co-expression of large and small subunits in E. coli results in no active holoenzyme being formed, suggesting that inappropriate folding of the large subunit may have occurred before the small subunit was able to bind. The difficulty in expressing higher plant Rubisco in a suitable host has made it difficult to engineer improved variants of the enzyme.
For these and other reasons, there exists a need for improved methods for producing plants and agricultural photosynthetic microbes with improved variants of enzymes involved in carbon fixation, for example, Rubisco.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention. All publications cited are incorporated herein by reference, whether specifically noted as such or not.